Tissue specificity of nuclear acidic proteins isolated from bovine brain and adrenal medulla.

Nuclear acidic proteins from bovine brain and adrenal medulla demonstrated dissimilar electophoretic and chemical profiles. The amino acid analysis of the acidic nuclear protein fraction isolated from these tissues revealed some variation in the ratio of acidic to basic amino acids. The estimation of free carboxylic acid groups confirmed the more acidic nature of the brain proteins. In comparing the acrylamide gels either visually or optically, several electrophoretically specific bands were apparent. Although the total number of protein bands from each tissue was approximately the same, the adrenal medulla contained a larger proportion of the more positively charged proteins. These observations are interpreted to indicate that the nuclear acidic protein from brain and adrenal medulla may show functional variation.     

Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.     

Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.     

The antibody from an infertile woman that induces sperm agglutination interacts with rat testicular cells and sperm

The serum obtained from an infertile woman induced a specific head-to-head agglutination of human and rat sperm. The immunoglobulin G (IgG) fraction of the serum was obtained and found to interact with the proteins of rat sperm in testis and epididymis. Using an indirect immunofluorescent method with rat sperm from vas deferens, we determined that the antibody recognized the protein on the convex and concave regions of the acrosome and over the entire tail. However, with testicular spermatozoa, the antibody recognized only the distal end of the tails. In paraffin sections of adult rat testis, sperm tails located at the luminal region of the seminiferous tubules stained intensely. Weak but significant staining also occurred on late spermatids. In the epididymal sections, staining was restricted to spermatozoa in the lumen. On the other hand, sections of testes from 25-day-old rats containing spermatogonia and early spermatocytes had a completely negative reaction. Testicular somatic cells, including Sertoli cells, peritubular myoid cells and interstitial cells, did not stain. To identify the testicular protein interacting with the antibody, adult rat testis proteins were prepared and analyzed by a sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblot technique. The antibody interacted with a protein with an estimated molecular weight of 82,000 in the testicular homogenate and particulate fraction, whereas the reaction was considerably weaker with the testicular cytosol fraction.     

Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa

We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.     

Localization of the antigotensin-converting enzyme activity in testis and epididymis

Angiotensin-converting enzyme (ACE) has been studied in different reproductive organs of the male rat, in somatic cell lines clonally derived from both rat and mouse testes, and in isolated spermatogenic cells of the mouse. Among the various reproductive organs only testis and epididymis show high levels of enzyme activity. The testicular activity is found mainly in the isolated germinal cells and residual bodies, whereas somatic cell lines contain negligible levels of activity even after addition of hormones and growth factors. Testicular homogenates, spermatogenic cells, epididymal spermatozoa, and spermatozoan cytoplasmic droplets, when fractionated by anion exchange chromatography, contain one major and one minor activity peak, whereas epididymal homogenates and epididymal secretions reveal an additional major activity peak together with the minor peak. All forms of ACE have a similar response to different modifiers, and are equally sensitive to the specific inhibitor N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl-L-proline (Enalapril). The testicular enzyme could provide a useful marker for spermatogenic maturation and/or cytoplasmic processes both in testis and epididymis. The separate epididymal peak is a secretory enzyme that may be responsible for the processing of spermatozoan plasma membrane constituents during epididymal transit, or may have a role in attacking some biologically active compounds.     

Isolation and purification of calcium-binding protein from electroplax of Electrophorus electricus.

An acidic calcium-binding phosphoprotein has been isolated from a cholinergic tissue, electroplax from Electrophorus electricus. The purification procedures included (NH4)2SO4 fractionation, boiling treatment, ECTEOLA-cellulose chromatography, and gel filtration on Sephadex G-100. Experiments were performed to compare this protein and a calcium-binding protein isolated from mammalian brain, adrenal medulla, and testis. These experiments showed that the two proteins were identical in terms of molecular weight (14 000), calcium-binding dissociation constant (kd=2.1-10(-5) M), electrophoretic mobility at pH 8.7 in 15% polyacrylamide gels, and phosphorus content (1 mol phosphorus per mol protein). In addition, the two proteins had similar amino acid compositions and peptide maps. Although the electroplax protein was not present in eel skeletal muscle, preliminary experiments indicated that small amounts of the protein were present in other eel tissues, namely brain, liver and spleen. These results suggest an identity between the electroplax and mammalian calcium-binding proteins and extend the findind of comparatively large amounts of the protein from mammalian nervous tissue to a cholinergic nervous tissue, electroplax. The close similarity of the proteins suggests a conservation of structure during evolution which may be required to fulfill a role in neuronal function.     

Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

Distribution of endogenous and exogenous 5′-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.     

Secretory granules from different glands contain neurocuprein-like protein

Secretory granules obtained from bovine pituitary, atrium and adrenal medulla contain an extremely acidic copper protein resembling by its main physico-chemical and antigenic properties as well as by the ability of its apoform to inhibit dopamine beta-monooxygenase the protein from brain, neurocuprein.     

Effects of osmolality, bicarbonate and buffer on the metabolism and motility of testicular, epididymal and ejaculated spermatozoa of boars.

Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.     

Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.     

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla

Neurocalcin is a novel calcium-binding protein found in bovine brain tissue. We investigated immunoreactivity for neurocalcin in the mouse adrenal medulla using light and electron microscopy. The immunoreactivity was present in nerve fibers, nerve terminals, and ganglion cells in the adrenal medulla, but chromaffin cells, sustentacular cells, and Schwann cells were negative in reaction. Nerve bundles containing neurocalcin-immunoreactive fibers passed through the adrenal cortex and extended into the medulla. Immunopositive nerve fibers branched off and projected varicose terminals around the chromaffin cells. These varicose terminals contained small and large-cored vesicles and made synapses with the chromaffin cells. We performed paraformaldehyde-induced fluorescence-histochemical studies for catecholamine combined with immunohistochemical studies for neurocalcin. Neurocalcin-immunoreactive nerve terminals were more abundant at noradrenaline (fluorescent) cell-rich regions than at adrenaline (non-fluorescent) cell-rich regions. These results show that neurocalcin-immunoreactive nerves mainly innervate noradrenaline-containing chromaffin cells in the mouse adrenal medulla and that neurocalcin may regulate synaptic function in the nerve terminals. Received: 21 October 1996 / Accepted: 12 February 1997     

Glycoproteins of ram sperm plasma membrane. Relationship of protein having affinity for Con A to epididymal maturation

Con A Receptors from the sperm plasma membrane were quantitated (using ³H acetyl-Con A) along the epididymal duct; they diminished in the second part of the epididymis as compared to the epididymal head. Glycoproteins having affinity for Con A were partially characterized: washed spermatozoa from rete testis (= testicular spermatozoa), middle corpus and distal cauda epididymis were labelled (¹²⁵I Na). Proteins of their plasma membrane were extracted (Triton ×100, 0.1% and chromatography affinity): differences appeared in ACA44 profiles from ¹²⁵I Con A Glycoprotein extractions between testicular spermatozoa (2 major peaks Kav= 0.41 and 0.52) and epididymal spermatozoa (3 major peaks Kav= 0.33–0.34, 0.41 and 0.52 and additional minor peaks between 0.66 and 1.00). The peak Kav= 0.41 diminished considerably on epididymal spermatozoa as compared to testicular spermatozoa.     

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida

Glioma pathogenesis‐related 1‐like protein1 (GliPr1L1) was identified by liquid chromatography‐tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine‐rich secretory proteins, Antigen 5 and pathogenesis‐related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N‐glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N‐glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti‐GliPr1L1 showed that this protein is involved in sperm–zona pellucida interaction. J. Cell. Physiol. 227: 3876–3886, 2012. © 2012 Wiley Periodicals, Inc.     

Changes in the protein composition of rat spermatozoa during maturation in the epididymis

Spermatozoa from the testis and cauda epididymidis were solubilized by detergent treatment and electrophoresis on SDS polyacrylamide gels revealed that the relative amounts of 13 detergent-extractable proteins decreased during passage of spermatozoa through the epididymis, 6 increased, whilst the remainder showed little or no change. Lactoperoxidase-catalysed iodination of plasma membrane proteins showed that the components carrying most of the label in testicular spermatozoa had Mr values of 110 000, 94 000, 84 000, 55 000 and 42 000 whereas on cauda epididymal spermatozoa the Mr values were 47 000, 24 000, 17 000, 14 500 and 13 500. Substantial differences were also noted in the protein composition of rete testis fluid and cauda epididymal plasma. The results support the concept that there is a considerable reorganization of the molecular architecture of the plasma membrane of spermatozoa during maturation in the epididymis.     

Peptidylglycine alpha-amidating monooxygenase from bovine heart atrium secretory granules and adrenal chromaffin granules

About 40-60% of the peptidylglycine alpha-amidating amonooxygenase activity in the lysates of secretory granules from bovine atria and adrenal medulla isolated and lyzed in the presence of pepstatin, phenylmethylsulfonyl gluoride, N-ethylmaleimide and catalase, was found to be in the soluble form. The remaining part bound to the membrane fraction was extracted with Triton X-100. The procedure of purification of the soluble form of peptidylglycine alpha-amidating monooxygenase from both atrial and chromaffin granules in electrophoretically homogeneous enzyme preparations was developed. The enzyme is made up of a single subunit with a molecular mass of 68 kDa and contains one copper atom per molecule. The EPR spectra of peptidylglycine alpha-amidating amonooxygenase and dopamine beta-monooxygenase were found to be practically identical, thus indicating that the copper environment in the both enzymes is the same. Both peptidylglycine alpha-amidating monooxygenase and dopamine beta-monooxygenase are inhibited by the neurocuprein apoform, an extremely acidic protein isolated from brain and secretory granules of different endocrine tissues.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

Monoamine Oxidase in Rat and Bovine Endocrine

Monoamine oxidase (MAO) was characterized in tissue homogenates from rat pancreatic islets, rat neurohypophysis and adenohypophysis, and rat and bovine adrenal medulla and adrenal cortex. Phenylethylamine was preferentially deaminated by rat pancreatic islet and bovine adrenal medulla MAO and with slight preference by rat neurohypophysis MAO, whereas 5-hydroxytryptamine was preferentially deaminated by MAO from all other endocrine tissues. Tyramine was a good substrate for all tissues. Clorgyline, a selective inhibitor of MAO-A, preferentially inhibited deamination of 5-hydroxytryptamine by all tissue homogenates, whereas deprenyl, a selective inhibitor of MAO-B, preferentially inhibited deamination of phenylethylamine. Km values for 5-hydroxytryptamine and tyramine were higher by one to two decimal powers than for phenylethylamine in homogenates from all endocrine tissues. Km values were significantly lower for 5-hydroxytryptamine and significantly higher for phenylethylamine in rat and bovine adrenal cortex than in adrenal medulla. According to these results, the contributions of MAO-B to total enzyme activity were 70% for rat pancreatic islets, 45% for rat neurohypophysis, 15% for rat adenohypophysis, 20% for rat adrenal medulla, 10% for rat adrenal cortex, 60% for bovine adrenal medulla, and 20% for bovine adrenal cortex. PC 12 cells also contained predominantly MAO-A (90%); however, an increased Km for phenylethylamine and a sensitivity of deamination of this MAO-B substrate to inhibition by clorgyline are indicators of abnormal behavior of MAO in this clonal rat pheochromocytoma cell line.